Among potential targets for antiviral therapy that arise during certain viral infections are the virus-coded proteinases. These enzymes, essential for the synthesis of infectious virus, are required to process virus-specific precursor proteins involved in the maturation, assembly and replication of such pathogenic human viruses as adenovirus, poliovirus, encephalitis virus, hepatitis A and C viruses, cytomegalovirus, and human immunodeficiency virus. virus-coded proteinases are highly specific for their virus-coded substrates. If equally specific inhibitors can be developed and targeted to infected cells, they should interfere with virus replication and not with normal cellular metabolism. Our model system is the infection of HeLa cells in culture by human adenovirus serotype 2 (Ad2). We showed for maximal Ad2 proteinase activity in vitro, three components are required- the protein product of the adenovirus L3 23K gene, an 11 amino acid peptide (pVlc) that originates from the C-terminus of virion precursor protein pvI, and the viral DNA. The cofactors increase k(cat), 300-fold with pVlc and 6000-fold with Ad2 DNA as well. We solved the three-dimensional structure of the proteinase complexed with pVlc at 2.6 Angstrom resolution. The fold of the protein is unique. A putative active site contains a Cys-His-Glu triplet and oxyanion hole in an arrangement similar to that in papain. If that is the active site, this protein would represent a new class of cysteine proteinases and a new, fifth group of enzymes that contain catalytic triads. Here we propose to understand at the biochemical and structural levels how the activity of the adenovirus proteinase is regulated. We shall locate and characterize the active site, elucidate the mechanisms by which the two cofactors stimulate proteinase activity and characterize the enzyme during different times after infection. We have just shown that beta-actin can serve as a cofactor and will characterize that interaction with the enzyme in vitro and in vivo. Although important in themselves, the results of these experiments will also be used in a subsequent grant to design different types of proteinase inhibitors to act as antiviral agents.